Fig. 2

Cav-1b expression is modulated during virus infection, and Cav-1b knockdown leaves morphants susceptible to infection.

A) Quantitative RT-PCR results revealed fold changes in the expression levels of Cav-1b in infected embryos when compared to uninfected embryos. Zebrafish were exposed seven dpf to 1×10⁶ TCID₅₀/mL virus. Total RNA was isolated from at 12, 24, and 48 hours post infection and reverse transcribed to cDNA (n = 20 fish per time point). All expression values have been normalized to the zebrafish β-actin gene. Error bars represent SEM of three replicates. B) Zebrafish embryos that were injected with Cav-1b morpholino (MO) to knock down the expression of Cav-1b or control MO were infected 48 hpf with 1×10⁶ TCID₅₀/ml virus and monitored for mortality. Results are representative of three separate experiments. Statistical analysis (Wilcoxon test) of the Kaplan-Meier curve was performed (*, p = 0.008). C) Zebrafish embryos that were injected with Cav-1b MO to knock down the expression of Cav-1b or control MO were infected by static immersion 48 hpf with 1×10⁶ TCID₅₀/ml virus. The graph indicates that early in infection (0–12 hpi), there is no difference in viral burden between Cav-1b morphants and controls. However, by 24–48 hpi, Cav-1b morphants have a higher viral burden. Figure is representative of three experiments; error bars are standard error of the mean (*, p<0.05). D) Western blot showing efficacy of MO knockdown in zebrafish. Zebrafish embryos from Control and Cav-1b MO, and Control MO with SHRV infection were compared for cav-1b expression at the 72 hpf developmental stage. At this time, infected fish were 24 hpi. Membranes were re-probed with antibody against β-actin to control for protein loading.